Mutant Bacillus lentus DSM 5483 proteases are derived by the replacement of at least one amino acid residue of the mature form of the B. lentus DSM 5483 alkaline protease. The mutant proteases are expressed by genes which are mutated by site-specific mutagenesis. The amino acid sites selected for replacement are identified by means of a computer based method which compares the three dimensional structure of the wild-type protease and a reference protease.
Legal claims defining the scope of protection, as filed with the USPTO.
1. A computer based method for identifying amino acid sites in a target protein which affect the stability of the target protein comprising the steps of (1) inputting the three dimensional coordinates of said target protein into a computer; (2) generating a probe-accessible surface of said target protein by probing the internal and external surfaces defined by the three dimensional coordinates of said protein with an uncharged probe molecule having a radius of about 0.9 to about 2.0 , wherein said probe-accessible surface has an external surface the interior of which contains one or more probe-accessible internal cavities; and (3) identifying the amino acids which make up the boundaries of the internal cavities, wherein said amino acids comprise a set of sites which when mutated affect the stability of the protein.
2. The computer based method of claim 1 wherein said target protein is Bacillus lentus DSM 5483 alkaline protease.
3. A computer based method for identifying amino acid sites in a target protein which affect the stability of the target protein comprising the steps of (1) inputting the three dimensional coordinates of said target protein into a computer; (2) generating a probe-accessible surface of said target protein by probing the internal and external surfaces defined by the three dimensional coordinates of said protein with an uncharged probe molecule having a radius of about 0.9 to about 2.0 , wherein said probe-accessible surface has an external surface the interior of which contains one or more probe-accessible internal cavities; (3) aligning said three dimensional coordinates of said target protein and a reference protein by moving the three dimensional coordinates of said reference protein into the coordinate frame of said target protein; and (4) identifying an amino acid in said reference protein whose side chain lies outside said external surface of said probe-accessible surface or inside said internal cavities of said probe-accessible surface.
4. The computer based method of claim 3 wherein said target protein is Bacillus lentus DSM 5483 alkaline protease.
5. The computer based method of claim 3 wherein said reference protein is any protein for which a three dimensional structure is available which is homologous to the target protein.
6. The computer based method of claim 3 wherein said reference protein is thermitase.
7. The computer based method of claim 3 wherein said reference protein is subtilisin Carlsberg.
8. The computer based method of claim 3 wherein said reference protein is subtilisin BPN .
9. The computer based method of claim 3 wherein said reference protein is proteinase K.
10. A computer based method for affecting the stability of a protein comprising the steps of: (1) inputting the three dimensional coordinates of said protein into a computer; (2) generating a probe-accessible surface of said protein by probing the internal and external surfaces defined by the coordinates of said protein with an uncharged probe molecule having a radius of about 0.9 to about 2.0 , wherein said probe-accessible surface has an external surface the interior of which contains one or more probe-accessible internal cavities; (3) identifying the amino acids which make up the boundaries of the internal cavities, wherein said amino acids comprise a set of sites which when mutated affect the stability of the protein; (4) identifying an amino acid mutation which changes the volume of said internal cavities; (5) determining if said amino acid in said protein can be mutated without creating unacceptable steric interactions; and (6) replacing the amino acid in said protein by site directed mutagenesis of the gene which expresses said target protein.
11. The computer based method of claim 10 wherein said target protein is Bacillus lentus DSM 5483 alkaline protease.
12. The computer based method of claim 10 wherein said reference protein is any protein for which a three dimensional structure is available which is substantially homologous to the target protein.
13. The computer based method of claim 10 wherein said reference protein is thermitase.
14. The computer based method of claim 10 wherein said reference protein is subtilisin Carlsberg.
15. The computer based method of claim 10 wherein said reference protein is subtilisin BPN .
16. The computer based method of claim 10 wherein said reference protein is proteinase K.
17. A computer based method for changing the stability of a target protein comprising the steps of (1) inputting the three dimensional coordinates of said target protein into a computer; (2) generating a probe-accessible surface of said target protein by probing the internal and external surfaces defined by the coordinates of said target protein with an uncharged probe molecule having a radius of about 0.9 to about 2.0 , wherein said probe-accessible surface has an external surface the interior of which contains one or more probe-accessible internal cavities; (3) aligning said three-dimensional coordinates of said target protein and a reference protein by moving the three dimensional coordinates of said reference protein into the coordinate frame of said target protein; (4) identifying an amino acid in said reference protein whose side chain lies outside said external surface of said probe-accessible surface or inside said internal cavities of said probe-accessible surface; (5) identifying the amino acid in said target protein which occupies the equivalent position as said amino acid in said reference protein; (6) determining if said amino acid in said target protein can be changed without creating unacceptable steric effects; and (7) replacing the amino acid in said target protein with the corresponding amino acid in the equivalent position in said reference protein by site-directed mutagenesis of the gene which expresses said target protein.
18. The computer based method of claim 17 wherein said target protein is Bacillus lentus DSM 5483 alkaline protease.
19. The computer based method of claim 17 wherein said reference protein is any protein for which a three dimensional structure is available which is homologous to the target protein.
20. The computer based method of claim 17 wherein said reference protein is thermitase.
21. The computer based method of claim 17 wherein said reference protein is subtilisin Carlsberg.
22. The computer based method of claim 17 wherein said reference protein is subtilisin BPN .
23. The computer based method of claim 17 wherein said reference protein is proteinase K.
Cooperative Patent Classification codes for this invention. Click any code to explore related patents in that topic.
May 31, 2000
June 11, 2002
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